D-Lactate dehydrogenase (D-LDH) of Escherichia coli is a membrane-bound, flavin-containing respiratory enzyme with a molecular weight of 65,000. It is one of the key dehydrogenases associated with the respiratory chain of E. coli. D-LDH catalyzes the oxidation of D-lactate to pyruvate, i.e., the transfer of 2 hydrogens (2 electrons and 2 hydrogen ions) from its substrate, D-lactate, first to its cofactor, flavin adenine dinucleotide (FAD), to form FADH2, which is then reduced by an electron carrier, ubiquinone, with the formation of ubiquinol. Ubiquinol is oxidized by the ubiquinol oxidases, which in turn reduce molecular oxygen to water. The proposed research represents our attempt to understand at the molecular level the relationship between the structure of D-LDH and its function, i.e., its interactions with its cofactor FAD and with other membrane components of the respiratory chain. In our research we the methodologies of biochemistry, molecular biology, stopped-flow kinetics, nuclear magnetic resonance (NMR), and electron paramagnetic resonance (EPR). We have constructed a plasmid in which the gene for D-LDH is under the control of the P(L) promoter. Upon temperature induction, this plasmid can express D- LDH at levels 300-fold over the wild-type cell, i.e., up to 35% of the total cell protein. Thus, our proposed approach to understanding the relationship between the structure and function of D-LDH is very advantageous because we have the ability to prepare specific mutants of D- LDH and to incorporate various nuclei (such as 2H, 13C, 15N, and 19F) by specific isotopic labeling into the wild-type and mutant D-LDHs, substrates, inhibitors, FADs, and ubiquinones needed for our research. In particular, we are interested in the structural features of the catalytic, cofactor, and lipid-binding domains and their relationship to the function of D-LDH. Ultimately we would like to know the molecular basis for the transfer of hydrogens and electrons upon the oxidation of D-lactate, catalyzed by D-LDH, from the catalytic site to FAD and then to ubiquinone. We believe that our proposed research will shed light not only on the molecular pathway in the respiratory process but also on the molecular basis of signal transduction phenomenon in biological systems.